Cloning of Aspartic Proteases from Mature and Immature Flowers of Silybum marianum (Asteraceae) with milk Clotting activity

COLOMBO, M.L.; LUFRANO, D.; VAIRO CAVALLI, S.E.

Mar del Plata

Congreso; XXIX Reunión Argentina de Fisiología Vegetal; 2012

Sociedad Argentina de Fisiología Vegetal

The aim of the present work was to clone the full-lenght cDNA encoding aspartic peptidase responsible of the milk clotting activity previously characterizedpresent in flowers of Silybum marianum (L.) Gaertn. (Asteraceae). Total RNA isolated from fresh flower buds was used as template in the reverse transcription PCR and full lenght zymogen cDNAs were further amplified by PCR using primers designed from conserved regions of the ORF 5´ and 3´ ends of plant AP. The 1500 pb amplified product was introduced into the pGEM-T Easy vector (Promega), cloned in XL1-Blue E. coli strain and submitted to automated sequencing. The c-DNA-derived amino acid sequence presented all the conserved features of typical plant AP and shows similarity with cyprosins and cardosins, the aspartic peptidases present in flowers of the well-known vegetable rennet C. cardunculus.